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Evolution of races within Fusarium oxysporum f.sp. lycopersici

Vadakkemukadiyil Chellappan, B.

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Citation for published version (APA):Vadakkemukadiyil Chellappan, B. (2014). Evolution of races within Fusarium oxysporum f.sp. lycopersici.

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Download date: 15 Jan 2020

General introduction

Chapter 1

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Introduction

Microorganisms are found everywhere, in the air, in water and in soil. The majority

of them is harmless, and some are even beneficial. For instance, specific

microorganisms are being used in biotechnology e.g. for the production of

metabolites and enzymes. Others are used as bio-control agents or bio-fertilizers1-3. In

contrast, pathogenic microorganisms are able to cause disease in humans, animals

and plants. Plant pathogens receive relatively much attention because they are a threat

to global food security by affecting crops. In the past, plant diseases have led to

failures of harvests with very serious consequences. Examples are the Irish Famine

due to late blight of potato (1845), the Coffee Rust epidemic in Sri Lanka (1875) and

the Bengal Famine due to brown spot of rice in India (1943). Plant pathogenic

microorganisms include viruses, bacteria, oomycetes and fungi. Fortunately, most

plant pathogens are capable of infecting one or a few host species only.

Their innate immune system helps plants to protect themselves against most

of the potential pathogens surrounding them. This immune system consists of two

layers4-6. The first layer is activated by the recognition of conserved pathogen-

associated molecular patterns (PAMPs) by corresponding pattern recognition

receptors (PRRs) of the plant7,8. This response is called PAMP-triggered immunity

(PTI). Examples of PAMPs are bacterial flagellin and elongation factor Tu (EF-Tu),

fungal ethylene-inducing xylanase (EIX) and oligosaccharides, such as glucan and

chitin fragments from fungal cell walls, and more complex epitopes such as

glycoproteins from oomycetes or bacterial lipopolysaccharides and peptidoglycans9-

12. Examples of PRR are the bacterial flagellin receptor FLS2, the EF-Tu receptor

EFR, and the chitin-binding receptors CEBiP and CERK112-16. Although PTI is

effective against a broad spectrum of microorganisms, pathogens overcome PTI by

secreting so-called effector proteins that manipulate cellular processes in the host to

facilitate susceptibility11,17-20. In turn, plants have evolved a second layer of immunity

in which they employ another type of receptors called resistance (R) proteins. R

proteins recognize specific pathogen effectors or their effects on the plant cell,

Introduction

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resulting in effector-triggered immunity (ETI)21,22. Effector proteins in the pathogen

that are recognized by specific R proteins in the host are called avirulence proteins

(Avr)23,24. The interaction between an R protein and its cognate Avr protein leads to a

!Figure 1. Zig-Zag model for the evolution of plant defense against pathogens (adapted from Jones and Dangl 200625). According to this model, the first line of active plant defense is triggered upon recognition of conserved molecules in the pathogens called pathogen associated molecular patterns (PAMPs, indicated in blue colour) by plants cell surface receptors called pattern recognition receptors (PRRs). This is called PAMP-triggered immunity (PTI). Successful pathogens overcome PTI by employing new secreted effectors (indicated in red colour) that suppress PTI responses, resulting in effector-triggered susceptibility (ETS). In turn, during evolution, plants have responded to these effectors through the development of resistant (R) proteins (indicated by black pie) that recognize single effectors, resulting in a second line of plant defense called effector-triggered immunity (ETI). The effectors that are recognized by R proteins are referred to as avirulence proteins (Avr) (indicated by red circlcs). Typically, the recognition of an Avr protein by an R protein is associated with programmed cell death, called the hypersensitive response (HR) that prevents the further growth of the pathogen in the host. As to counteract, pathogens have evolved to overcome ETI by the loss of function of its avirulence proteins or by employing new effectors (indicated by green circles) to suppress ETI. In turn, plants have evolved new R proteins (indicated by pink pie) to recognize newly evolved pathogen effectors, resulting again in ETI.

!

disease resistance response, often a so-called hypersensitive response (HR), a

programmed cell death at the site of infection site by which further growth of the

pathogen in the plant is restrained8,25. In response to this, pathogens may overcome

Chapter 1

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ETI by loss-of-function of the avirulence protein or by employing new virulence

factors. In the plant, new R proteins may evolve that recognize other pathogen

effectors. This leads to a molecular arms race between the host plant and its pathogen,

which is illustrated in a Zig-Zag model (Figure 1)25. This type of molecular arms race

can be seen in many plant-pathogen interactions. A good example is the interaction

between tomato and its vascular wilt pathogen Fusarium oxysporum f.sp.

lycopersici26.

Fusarium oxysporum

Fusarium oxysporum is a (predominantly) asexual soil-borne fungus occurring

worldwide. The species harbors both pathogenic and non-pathogenic strains. Because

of the widespread presence of non-pathogenic isolates, it is generally assumed that

pathogenic strains emerged from nonpathogenic ancestors27. Pathogenic forms may

cause devastating diseases on several economically important crops28. Recently, F.

oxysporum has been reported to be a serious emerging human pathogen causing

invasive fungal infection with lethal outcomes in immuno-compromised patients29,30.

Strains pathogenic to plants often cause vascular wilt diseases but can also cause

damping-off problems or crown and root rot31-33. Although they have a wide host

range, individual strains of F. oxysporum infect one or few plant species only. Hence,

based on their host specificity, F. oxysporum strains are divided into formae speciales

(special forms)34. More than 120 formae speciales of F. oxysporum have been

described35. For example, F. oxysporum f.sp. lycopersici (Fol) causes disease only in

tomato (Solanum lycopersicum L.). However, some formae speciales have broader

host ranges, such as F. oxysporum f.sp. radicis-cucumerinum and F. oxysporum f. sp.

radicis-lycopersici, which apart from infecting cucumber and tomato, respectively,

can cause root and stem rot on other host plants36. As a (predominantly) asexual

organism, F. oxysporum is thought to evolve by means of clonal mutations at

virulence or avirulence loci and potentially through the process of parasexual

recombination37. Heterokaryon formation is a prerequisite for parasexual

recombination and is limited to strains that are vegetatively compatible. Strains that

are able to form heterokaryons with one another are assigned to the same vegetative

Introduction

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compatibility group (VCGs)27,38. In Fusarium spp. and other fungi, vegetative

compatibility is controlled by vegetative (vic) or heterokaryon (het) incompatibility

loci39. Two fungal strains are vegetatively compatible only if they possess identical

alleles at corresponding vic loci. Therefore, strains belonging to the same VCG are

genetically very similar. VCGs have often been used to estimate the diversity within

the formae speciales of F. oxysporum. However, a mutation at a single individual vic

locus would cause incompatibility between closely related individuals, grouping them

into different VCGs40. Therefore, the genetic relationships between VCGs must be

assessed in other means. Many formae speciales of Fusarium oxysporum are

comprised of multiple VCGs, suggesting that the pathogenicity to a particular host

has evolved more than once41,42. Most formae speciales of F. oxysporum include two

or more races. Commonly, the relationship between formae speciales, races and

VCGs is complex. Within certain formae speciales, multiple races may occur in

single VCG and the same race may be present in multiple VCGs.

F. oxysporum f.sp. lycopersici

Fol is the causal agent of Fusarium wilt of tomato, first described by Massee in

189543. Three physiological races of (Fol) have been reported based on their ability to

infect tomato cultivars carrying different monogenic resistance genes. During the first

half of last century race 1 was the most prevalent race. Resistance to this race was

described first by Bohn and Tucker in 193944. Soon after the introduction of the

resistance into commercial tomato varieties, a Fol race (race 2) was identified

capable of breaking the resistance. Race 3, capable of breaking both race 1 and race 2

resistance was observed in Australia in 197845. To date, all Fol races have been

reported from many if not all tomato-growing countries46-49. So far, four VCGs

(VCG0030, VCG0031, VCG0033 and VCG0035) of Fol have been reported47,48,50.

Strains formerly grouped in VCG0032 are now included in VCG003051. VCG0030

includes all races, VCG0031 includes races 1 and 2 only, and VCG0033 and

VCG0035 only include race 3 and race 2, respectively. Previously, results from

restriction fragment length polymorphisms (RFLP), random amplified polymorphic

DNA (RAPD) and isozyme analysis demonstrated that races within a VCG are

Chapter 1

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genetically more similar each other than to isolates of the same race in another VCG,

suggesting that VCG is an indicator of the evolutionary lineage of Fol49,51,52.

Similarly, a phylogenetic study based on the ribosomal DNA intergenic spacer (IGS)

region have revealed three evolutionary lineages (in a study in which VCG0035 was

not included) for Fol in which each lineage comprised of isolates belonging to a

single VCG53. The occurrence of multiple races in each lineage suggested that races

have evolved independently within each lineage.

Pathogenesis

The mycelium of Fol is floccose, sparse or abundant and ranges in color from white

to pink. It produce three types of spores; macroconidia, microconidia and

chlamydospores. Macroconidia are falcate and thin walled, and have three or four

septa. They are borne on branched conidiophores in sporodochia. Microconidia

usually are non-septated, oval-elliptical, and are formed abundantly in false heads on

short phialides formed on the hyphae. Chlamydospores are thick walled and are

formed abundantly in hyphae. When food becomes scarce, Fol can survive for a

longer period of time in soil as chlamydospores. Spores are dispersed by many

different means including wind and through seeds, or infected planting material. The

infection process of Fol in tomato can be divided into several stages: root recognition,

root surface attachment and colonization, penetration and colonization of root cortex

and hyphal proliferation within the xylem vessels35. In the soil, spores are stimulated

to germinate by exudates from roots growing nearby, and the newly developing

hyphae show a positive tropic response to roots and root surfaces54. Root penetration

occurs through natural wounds at the origin of lateral roots, or by direct penetration

of the cortex. After entry of the cortex, the pathogen colonizes the xylem vessels and

rapidly spreads throughout the plant. Plants respond to Fol infection by secreting

several antimicrobial compounds and proteins such as glucanases, chitinases and

other pathogenesis related (PR) proteins into the xylem vessels, and by the formation

of tyloses, gels and gums. As a consequence of the latter, xylem vessels are blocked

preventing further growth of the pathogen in the plant55,56. In susceptible tomato

plants, the fungus keeps proliferating in the vessel tissue and the resulting occlusion

Introduction

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of an ever increasing number of xylem vessels results in a reduced water flow and

consequently in wilting and eventually death of the plant (Figure 2A). At this stage,

F. oxysporum spreads to the neighboring parenchymatous tissue and starts

sporulating on the plant surface, thereby completing its pathogenic life cycle28. Since

Fol kills the plant at least at later stages of infection, it is generally considered a

hemibiotrophic pathogen.

!!!!!!!!!!!!! !! Figure 2. Response of a susceptible tomato cultivar and a resistance tomato cultivar against Fol infection. A) General susceptible tomato line infected by Fol race 1. Ten days old tomato seedlings (cultivar moneymaker C32) were inoculated with a Fol race 1 isolate B) Tomato cultivar carrying I resistance gene (cultivar GCR161) show resistance against Fol race 1. Both pictures were taken three weeks after inoculation.

Resistance genes in tomato and corresponding avirulence genes in Fol

For tomato and Fol, a gene-for-gene relationship exists and tomato R genes and

corresponding avirulence genes in Fol have been identified. Monogenic resistance

genes against Fol, called immunity (I) genes, have been identified in wild tomato and

introgressed into tomato cultivars57,58. R genes I and I-1 correspond to AVR1 (SIX4)

and confer resistance against Fol isolates carrying this avirulence gene (Figure 2B). I-

2 and I-3 recognize Avr2 (Six3) and Avr3 (Six1), respectively, and confer resistance

against Fol isolates carrying the genes encoding these effectors. So far only I-2 and I-

3 have been cloned59,60. I-2 is predicted to encode an intracellular protein from the

CC-NB-LRR family and I-3 encodes SRLK receptor like kinase59. NB-LRR proteins

are commonly involved in the recognition of effectors from bacteria, viruses, fungi,

oomycetes and nematodes. AVR1 (also known as SIX4) is present only in race 1

!!!!!!!!!!!!!!!!!!!!!!!!!!!!(!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

Chapter 1

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whereas AVR2 and AVR3 are present in all Fol races (Table 1). However, AVR2 is

mutated in race 3: three alternative nucleotide substitutions have been found (G121A,

G134A and G137C) that cause loss of its avirulence function 61. Besides their

avirulence function, Avr2 and Avr3 have been shown also to function as virulence

factors, as deletion of their genes compromises virulence of Fol on general

susceptible tomato plants61,62. Although Avr1 is not a virulence factor, it has been

shown to suppress the I-2- and I-3-mediated resistance in tomato cultivars. This

suppressive function of AVR1 enables race 1 isolates to infect I-2 and I-3-containing

tomato cultivars despite the presence and expression of AVR2 and AVR363.

Table 1: Relationship between Fol races and tomato cultivars

“-“ - Absence of AVR1, “x” – Some race 1 isolates are virulent on I-2 and I-3 lines51,63,66 (Chapter 2), “y” – allele containing a point mutation that prevents recognition by I-261.

Genome organization of Fol

The genome of Fol isolate 4287 (Fol4287) has been sequenced, assembled and

annotated, and is publically available (www.broadinstitute.org). Fol4287 is a race 2

isolate and belongs to VCG0030. Although not assembled, the genome sequence of

another Fol isolate, race 3 isolate MN25 (VCG0033), is publically available as well

(www.broadinstitute.org). The availability of annotated Fol genomes provides an

excellent opportunity to explore the evolution of this fungus through comparative

studies. The genome size of Fol4287 has been estimated to be 61 Mb and is organized

into 15 chromosomes (and 117 unpositioned scaffolds). Comparison of this F.

oxysporum genome to those of other Fusarium species such as F. verticillioides, F.

Race Genotype Resisted by

1 AVR1, AVR2, AVR3 I, I-1, I-2x and I-3x

2 -, AVR2, AVR3 I-2 and I-3

3 -, avr2y, AVR3 I-3

Introduction

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graminearum and F. solani revealed four lineage specific (LS) chromosomes in Fol

that are rich in transposons, genes encoding effector proteins, transcription factors

and proteins involved in signal transduction; they are devoid of house-keeping

genes64,65. The 11 core chromosomes of Fol show a high level of synteny with

corresponding chromosomes of Fusarium verticillioides, indicating that LS

chromosomes may have a distinct evolutionary origin (Figure 3). Interestingly,

horizontal transfer of one LS chromosome, notably chromosome 14, from a

pathogenic isolate of Fol to a non-pathogenic isolate of Fo during co-cultivation,

resulted in a new pathogenic lineage infecting tomato, indicating that chromosome 14

carries the main determinants for the ability to cause disease in this plant species64.

Fol4287 chromosome 14 contains a number of genes encoding small, relatively

cysteine rich, secretory proteins collectively known as Six proteins as they are

secreted in the xylem61-65. The proteins encoded by most SIX genes on chromosome

14 have been identified in the xylem sap of Fol infected tomato plants and some of

them have been shown to be virulence factors that promote Fol infection on

tomato20,61-63,65-68. Some SIX genes appear to be unique to Fol (e.g. SIX3 and SIX5)

but others, including SIX4, SIX6, SIX7, SIX8 and SIX9 have close homologues in

other formae speciales20,68,69. The SIX genes are well conserved in Fol except for

SIX4, which is restricted to race 1 isolates only63,68.

In asexual fungi transposable elements (TE)-mediated mutations appears to be

the main source of genetic variability70. Recombination between (nearly) identical

transposable elements can create structural rearrangements like deletions, inversions,

duplications and translocations. For example, homologous recombination between

two occan transposable elements led to the deletion of three copies of avirulence gene

AVR-pia in the rice blast fungus Magnaporthe oryzae, enabling this fungus to infect

rice cultivars carrying corresponding resistance gene Pia71,72. Moreover, insertion of

TEs into an ORF or promoter region can also lead to the loss of function of

genes46,73,74. In addition, TEs may have roles in evolution related to their ability to

create novel genes. For example, Helitron TEs exhibit the remarkable ability to

capture gene fragments from multiple genomic location and in some cases, the

captured gene fragments fuse to form a chimeric transcript75,76. Helitrons are thought

Chapter 1

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to replicate via a rolling circle replication manner77. A recent study has revealed that

Fol possesses many different families of class I and Class II transposable elements,

including Helitrons, of which 74% are located in LS regions65.

Figure 3. Whole genome comparison between Fusarium verticillioides (Fv) and Fusarium oxysporum f.sp. lycopersici (Fol). Black lines indicate the core chromosomes and red lines indicate the lineage specific (LS) chromosomes and chromosome extensions in Fol.

Current concept of evolution of races in Fol

Based on the gene-for-gene relationship between races of Fol and cultivars of tomato,

a model for the evolution of Fol races in agriculture has been proposed26. According

to this model, the historically ‘oldest’ race 1 carries AVR1, AVR2 and AVR3 in its

genome. After the introduction in tomato cultivars of R gene I in 1939, race 2 isolates

evolved by loss of AVR1. Subsequently, after the introduction of I-2 in tomato

cultivars in 1965 race 3 evolved by loss-of-avirulence mutations in AVR2. I-3 was

introduced from Solanum pennellii in the late 1980s to protect tomato against race 3.

Since Fol race 1 isolates have the ability to suppress I-3-mediated resistance, this R

gene may not be fully effective against race 1. A combination of I-1 or I and I-3

should yield broad resistance of tomato to Fusarium wilt disease of tomato, since I-3

is directed against a virulence factor (AVR3) and I (and I-1) against the suppressor of

F. verticillioides F. oxysporum. f.sp. lycopersici

!

Introduction

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I-3 (AVR1).

Outline of the thesis

To unravel the molecular events underlying the evolution of Fol races, this thesis

mainly focuses on AVR1. The lack of I-2- and I-3-mediated resistance-suppressive

activity in some Fol race 1 isolates is investigated in Chapter 2. The analysis showed

that lack of suppression in some race 1 isolates cannot be explained by differences in

either the AVR1 coding sequence or its surrounding sequences nor by an altered

expression level. Based on the results shown in Chapter 2, I propose that lack of

suppression is due to the influence of an unknown genetic factor present in some race

1 strains of F. oxysporum f.sp. lycopersici

Chapter 3 investigated the molecular mechanism underlying the evolution of

Fol race 2 from race 1. Using a BAC library of race 1 isolate Fol004, a 100 kb

genomic region containing AVR1 has been sequenced and annotated. The evidences

obtained by comparing this AVR1 genomic region to the sequenced genome of

Fol4287 showed that Fol race 2 evolved from race 1 by deletion of a genomic

fragment containing AVR1, most likely due to a homologous recombination event

between two Helitron transposable elements bordering this fragment.

Helitrons form a recently discovered class of transposons that occur in a wide

range of eukaryotes including plants, animals and fungi. In plants and animals, the

characteristics of Helitrons are well documented. In fungi, the occurrence and

characteristics of Helitrons have hardly been investigated. In Chapter 4, a novel

family of Helitrons in F. oxysporum (Fo), designated FoHeli, with distinct terminal

structural features compared to plant and animal Helitrons is described. Phylogenetic

analyses of the rep and hel domains, hallmarks of Helitrons, reveal that different

FoHeli groups arose through ancient duplications in the Helitron family and that

FoHeli’s with similar structural features cluster in one clade, indicating that the

evolution of these features occurred only once.

Chapter 1

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In Chapter 5, the evolution of Fol races is investigated in more detail. So far,

three point mutations in AVR2 gene leading to the evolution of race 3 from race 2 had

been documented. In this study two novel mutations in Avr2 are described that also

lead to the evolution of race 3 from race 2. A phylogenetic tree based on EF1-!

sequences from all Fol races used in this study shows five clonal lineages that

correlate with known VCGs. The analysis showed that Fol races likely emerged

within VCGs independently at different times. Finally, a model for the evolution of

races within Fol is proposed.

In Chapter 6, the results described in this thesis are summarized, discussed

and a model for the origin of formae speciales within F. oxysporum and evolution of

races within formae speciales is proposed.

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